RNAguy123

Sorry I was unclear, I meant one band for each run (since I did it in duplicate there were 2 bands total). But each loaded lane had just one distinct band corresponding to a 22mer. My sample is a desalted RNA oligo from Sigma, I'm just trying to make sense of the detreioration of signal in the mass spec. Thanks again CharonY

Thanks again for all the replies. I am going to provide an update. I ran a 15% PAGE in duplicate with my sample, and two distinct bands showed up right as expected for a 22mer of this size indicating degradation is not the problem (or at least the sample is not yet degraded so perhaps it was at the source, although two weeks prior I ran the sample with identical conditions and had great results). So I guess I remain baffled.

Thanks a lot for the suggestions. I will look into cryoprotectants and definitely will use buffer solutions now. I appreciate the insight! Got it CharonY, thanks a lot it makes sense. I will definitely run a gel and see what sort of state my sample is in, thanks for the suggestion.

Thanks very much for your reply. I agree I do think it is degradation as well and my fault for improper storage, I just don't get the resulting mass spec, shouldn't I see an array of fragments for degraded RNA and not one sharp peak at m/z=265? My sample can adopt a maximum charge of -24, however unlikely to see that high of a charge state following ESI. I do direct injection and then can separate afterward depending on m/z. Again I do agree with you in thinking it is degradation, I am just trying to make sense of the resulting mass spectrum. Thanks again CharonY

Hey all, So I have a sample of RNA with a MW of ~7000 g/mol forming a stem-loop structure, and about a month ago I used ESI to run the sample on the mass spec and saw the expected distribution of charge states cleanly (sprayed from methanol:water in the negative mode). I stored the RNA sample in MQ water at -20 celsuis in the meantime. Now I tried again today using the same ESI conditions with my sample and saw nothing new to the background except for a peak at m/z=264 or 265. There is nothing in the upper ranges of m/z=600-1000 which I was looking successfully at a month ago. Now my first guess is the RNA has degraded during storage, but what I don't get is wouldn't I see a distribution of fragments and not just a single clean peak at m/z=264? The peak was not present in the solvent background alone so I have no idea what it could be. Any insight would be much appreciated! Thanks a lot